Sunday, June 24, 2007


Invetigator: Rupa Singh

Proteases form a large group of enzymes belonging to the class of Hydrolases, ubiquitous in nature and occupy a major role with respect to their application in both physiological and commercial fields. They are molecules of relatively small size and are compact, spherical structures that catalyze the cleavage of peptide bonds in proteins. Based on the functional group present at the active site and their catalytic mechanism, proteases are classified into four groups: serine proteases, aspartic proteases, cysteine / thiol proteases, and metalloproteases (Jewell, 2000). Depending on their site of action, proteases are broadly divided into either exopeptidases or endopeptidases. Endopeptidases (like: trypsin, chymotrypsin, pepsin, papain) cleave the peptide bonds distant from the termini of a substrate, whereas exopeptidases (like: aminopeptidases, carboxypeptidases A) cleaves the peptide bond proximal to the amino or carboxy terminus of a substrate(Ryan, 1973).
Proteases enzymes are important in diverse and crucial biological processes; for example, they are involved in the regulation of metabolism and gene expression, enzyme modification, pathogenecity, blood clotting cascade, complement system, apoptosis pathways, invertebrate prophenol oxidase activating cascade, and the hydrolysis of large proteins to smaller molecules for transport and metabolism (Rao et al., 1998). Proteases are also used in medicine for several clinical studies showing their benefits in oncology, inflammatory conditions, blood rheology control, and immune regulation. Proteases are able to hydrolyze almost all proteins as long as they are not components of living cells. Normal living cells are protected against lysis by the inhibitor mechanism.
Human proteases are responsible for apoptosis, MHC class II immune responses, prohormone processing, and extracellular matrix remodeling important to bone development etc. and are also involved in the occurrence of diseases like tumor metastasis, rheumatoid arthritis and HIV AIDS. HIV-1 protease helps in the maturation of the core proteins and in the assembly of virus which is required for viral infectivity (Peng et al., 1989 ). But the invention of inhibitor of this protease has somewhat reduced HIV RNA levels and increased CD4+ lymphocyte counts (Kakuda et al., 1998).
Microbial proteases are highly relevant in technical enzyme application , and are most prominent with respect to market value and tonnage since they are extracellular enzymes .For instance, Subtilisins (a serine protease) are used as effective detergents to degrade proteinaceous stains, and typical stains of blood, milk, sauces (Maurer, 2004).
Plant proteases form a valuable reservoir of biomolecules. Plant latex proteases, being thermostable and resistant to the action of synthetic inhibitors, are predominantly applied in medicine for the dissolution of clots, debridement of wounds etc. and in industry for meat tenderization, detergency, dough conditioning, protein recovery from organic wastes. The role of plant aspartic proteases has been studied in plant senescence, programmed cell death (PCD), embryo and endosperm development, growth of pollen tube, proteolytic processing and maturation of storage protein, and defense against pathogens (Simões and Faro, 2004). These proposed biological function are still speculative, so more work is needed to confirm the proposed hypotheses or to elucidate new functions. Metalloprotease perform dual function in mitochondria and chloroplasts degrading their target peptides (Bhusan et al., 2003).
Choerospondias axillaries: (Locally called “lapsi”), is a large, deciduous fruit-bearing tree of the family Anacardiaceae. A native of the Nepal hills (850–1900 m); it has also been reported from India, China, Thailand, Japan and Vietnam. Lapsi wood is used as light construction timber and fuelwood; seedstones are used as fuel in brick kilns and the bark has medicinal value. Nepal is unique in processing and utilizing lapsi fruits. The fruits are rich in vitamin C content, and are consumed fresh or pickled and are processed into a variety of sweet and sour, tasty food products locally called Mada and candy. Though lapsi has commercial value, only few researches have worked on it. So, it provides a large platform to work on. (Agrawal et al,1992) found the interfering protease while studying the male related protein in lapsi and reported that its maximum activity is at 50°C.Besides, this thermostable protease showed its activity even in the presence of 2mM PMSF which eradicate the possibility of being a serine protease. (Dekhang and Sharma, 2006) reported that the crude protease has an optimum pH of 7.0 and retains considerable activity even at 70ºC. He also reported that PMSF has no effect on the activity and cysteine protease inhibitor iodoacetic acid sodium salt inhibits its activity to some extent. This protease is activated by 2-mercaptoethanol and don’t follow conventional exopeptidase mechanism.
Despite these research, this protease is not fully characterized. So this work will be directed towards isolation ,characterization,purification and then, commercialisation of this protease.

Extraction of protease from leaf.

Purification of the protease by ammonium sulfate precipitation, affinity chromatography .

Detection of its purity through SDS gel electrophoresis .

Determination of its proteolytic activity by Ninhydrin and TCA precipitation method.
Chemicals and Reagents:
Phosphate buffer, Trichloroacetic acid (TCA), ethanol, Bradford reagent, ammonium sulfate, ammonium persulfate, acetic acid, acrylamide, bis-acrylamide, Coomassie Brilliant Blue R-250, N,N,N',N'-Tetramethylene ethylenediamine (TEMED), Sodium dodecylsulfate (SDS), acetone, Equilibration buffer (0.1M pH7.0 phosphate buffer containing 2M amm.sulfate), Elution buffer (0.1M pH7.0 PB containing 1M,0M amm.sulfate respectively), Phenyl agarose.
Protein gel electrophoresis set, analytical balance, cold centrifuge, micropipettes, magnetic stirrer, UV/ Vis spectrophotometer, pH meter, water bath, Whatman No. 1 filter paper, vortex machine, dialysis tubing, 1cm-cuvette, Eppendorf tubes, chromatographic column

Preparation of cell free extract:
1. Green leaves of lapsi will be collected, dried in shaded area.
2. Dried leaves will be blended in a blender to get fine powder.
3. 1 gm of dried keaf powder
Preparation of crude extract:
1. Acetone powder will be homogenized in two volumes of 0.1M phosphate buffer, pH 7.0 containing 0.1% 2-ME (2mM) and 2 mM PMSF at room temperature.
2. The homogenate will be centrifuged at 5,000 rpm for 5 minutes.
3. An aliquot of the supernatant will be stored in freezer for subsequent analysis i.e. quantitation of total protein, access of protein purity by SDS-PAGE and proteolytic activity.
Ammonium sulfate precipitation: (Little and Little, 1994)
Solid ammonium sulfate will be added to the supernatant obtained from the above step to get 20% saturation (little at a time). The solution will be mixed for 30 minutes.
And will be Centrifuged for 10 minutes at 10000 rpm .Pellet will be dissolved into minimum volume of 0.1 M Phosphate buffer, pH 7.0 .And solid ammonium sulphate will be added to get 40% saturation to supernatant. Then will be centrifuged for 10 minutes at 10000 rpm.The above procedure will be repeated to get 60 and 80% saturation.
Dialyze, do SDS-PAGE, and assay for protease activity for each fractions of pellets.

The number of grams of ammonium sulfate to be added to achieve desired saturation:
g = 533(S2-S1)/100-0.3S2; where g is the amount of ammonium sulfate in grams added per 1 ltr of solution at 20°C; S1 is the initial % saturation and S2 is the final % saturation.

Ion exchange chromatography (, 17th June 2007):

This protease is assumed to be a cysteine protease (Dekhang and Sharma, 2006). So anion exchanger chromatography will be used to purify the fraction having highest activity of protease.
DEAE Sepharose, an anion exchanger resin will be used to prepare a 10 ml column. Required amount of DEAE Sepharose will be suspended in equilibration buffer for overnight to let the beads swell.
The slurry will then be added to the column with its outlet closed. After the beads have settled slowly the buffer will be let to flow out at a rate of 0.5 ml/ minute.
Equilibration buffer will be passed through the column 2-3 more times so as to pack the column smoothly and also to equilibrate it with buffer.
Sample protein will be added to the top of the column (without disturbing the top of the column). The column will be washed with equilibration buffer 3-5 times to remove extra proteins.
Then the protease will be eluted out using elution buffer. 1 ml per tube will be collected.
The protein concentration in each fraction will be determined by measuring absorbance at 280 nm and the fractions around peaks will be dialyzed. The dialyzed sample will then be subjected to SDS-PAGE and protease assay.
Further purification, if required, will be done by gel permeation chromatography.

Gel-Permeation Chromatography (Andrews P, 1965):
Slurry of Sephadex G50 in 0.15 M KCl will be prepared to get a bed volume of 50 ml. Required amount of gel beads will be mixed with KCl and the beads will be allowed to swell for 24 h.
Slowly, the swelled beads will be poured in the column with outlet closed. The beads will be left to settle and the KCl solution will be then passed through the column at a flow rate of 0.5 ml/ min. The column will be percolated with the same KCl solution 1-2 times so that column will be well packed.
The column will be equilibrated with 0.05 M Tris.HCl, pH 7.5 containing 0.1 M KCl.
The dialysed, partially purified protease sample will be applied on top of the column.
The protein will be eluted at a flow rate of 0.5 ml/ minute and 3 ml sample per tube will be collected.
Each eluted fraction will be quantitated by taking absorbance at 280 nm and the fractions will be pooled showing peak value.
The pooled fraction will be dialysed against 0.1 M Phosphate buffer, pH 7.0 containing 50% glycerol for 12 h with 2 changes of buffer.
The protein content will be determined, and then SDS-PAGE and assay will be done.

Dialysis tubing will be sealed at one end with a knot.
The protein fractions will be poured into the dialysis bag and the top end will also be sealed.
The bag will be immersed in a 300 ml of 0.1 M Phospahte buffer, pH 7.0 containing 50% glycerol and agitated gently with a magnetic stirrer.
Dialysis will be continued for 12 h with 2-3 changes of buffer.

Bradford assay for protein concentrations (Bradford, 1976):
Protein concentrations in various samples will be determined by Coomassie Brilliant Blue G-250 binding assay using bovine serum albumin as a standard. 0.1 ml of protein sample will be mixed with 4.9 ml of Bradford reagent and absorbance will be measured at 595 nm.

Assay for proteolytic activity (Agrawal et al., 1992):
BSA (10 mg/ml) 10μl will be incubated with 0.1 ml of enzyme solution for various time intervals viz 30sec, 1 min, 10 min at room temperature. After incubation, the reaction will be stopped by adding equal volume of 10% cold TCA and kept at -20ºC for half an hour. The non-degraded BSA will then be separated by centrifugation at 10,000 rpm for 10 minutes. The pellet obtained will be washed two times with equal volume of ethanol: ether (1:1) and dried at room temperature after which it will be dissolved in 100 μl of 0.1 N NaOH. Bradford assay will then be performed to determine the amount of degraded BSA.

Assay for proteolytic activity in the presence of SDS coupled with heat:
Enzyme solution (0.1 ml) will be mixed with different concentration of SDS and will be heated at temperatures 30, 40 and 50 °C. Then the protease activity will be accessed. Also, the activity will be accessed by first mixing enzyme, SDS and BSA and then heating.
SDS-PAGE (Laemmli, 1970):
Purity of sample will be assessed by SDS gel electrophoresis.
water trisHCl SDS Acrylamide Mixture (30%+ .8%)
Components : Stacking Gel (4%) 3.075 ml 1.25 ml.(0.5 M, pH 6.8) 0.025 ml. 0.67 ml
Resolving Gel (15%) 2.4 ml 2.5 ml.(1.5 M, pH 8.8) 0.05 ml. 5 ml.
Ammonium persulphate : 0.01 gm.
TEMED : 10 µl
Running buffer
Loading buffer
Staining solution
Destaining solution

Production of polyclonal antibody (Khatri et al., 2004):
a) Immunization:
1. The purified protease (antigen) will be dissolved in phosphate buffer (pH=7.0) for concentration
2. The antigen will be mixed with equal amount of Freund’s complete adjuvant and emulsified with 26G needle.
3. The rabbit will be injected intramuscularly in right flank.
4. Three booster doses with Freund’s incomplete adjuvant were injected with the interval of 14 days in opposite flank.

b) Serum preparation:
The blood will be obtained from the ear vein of immunized rabbit by using butterfly needle after 7 days of third booster dose.
The collected blood will be placed at 57°C for 30 minutes to inactivate complement.
The blood will be then placed at 4 °C overnight to clot.
The clotted blood will be decanted into the centrifuge tube and will be centrifuged at 4°C at 10,000rpm for 10 minutes.
The supernatant will be decanted in new tube and the serum will be stored at -20°C in aliquot volumes.

c) Monitoring antibody titer:
The quality and quantity of antibodies in serum (humoral immune response) of the bleeds will be monitored by indirect ELISA. And the antibody will be purified using ion exchange chromatography.

Flow chart for immunization schedule (Khatri,2002)

Antigen selection
(Purified protease, 1 mg/ml)

Shocking dose
(0.5 ml of protease+0.5 ml of Complete Freund's adjuvant (CFA),
Final protein concentration 500 µg/ml)

(Emulsification with 18G needle)

Check for stable oil in emulsion

Ist Booster dose
(Inject 450-500μg/dose with 26G needles emulsified with incomplete adjuvant in opposite flank to that of shocking dose)

IInd Booster dose
(Inject 450-500μg/dose with incomplete adjuvant in opposite flank to that of Ist Booster dose)

IIIrd Booster dose
(Inject 450-500μg/dose with incomplete adjuvant in opposite flank to that of IInd Booster dose)

Serum Collection after 7 days of IIIrd Booster dose

Store the antisera at -20°C in small aliquots (0.1 to 0.5 ml)

The crude extract from leaf of lapsi was assayed for its mechanistic class and proteolytic activity by previous researchers. Yet, no any confirmed results have been found, may be due to incomplete purification. But this time, complete purification of protease will be done so that the substances that mask the protease and hamper its activity will be completely removed.


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